High level expression and purification of recombinant human serum albumin in Pichia pastoris

Zhu, Wen; Gong, Guihua; Pan, Jie; Han, Song; Zhang, Wei; Hu, Youjia; Xie, Liping

doi:10.1016/j.pep.2018.02.003

Cited by 47 publications

(25 citation statements)

References 31 publications

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“…HSA production with Mut + or Mut S strains has already been attempted by some researchers, and by selecting for multicopy clones and optimizing the process, they obtained end titers of up to 10 g/L (Mallem et al, 2014; Zhu et al, 2018). As our work is still at a proof of concept stage, we used single copy clones and did not further optimize the process.…”

Section: Discussionmentioning

confidence: 99%

Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications

Zavec

Gasser

Mattanovich

2020

Biotech & Bioengineering

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The methanol utilization (Mut) phenotype in the yeast Pichia pastoris (syn. Komagataella spp.) is defined by the deletion of the genes AOX1 and AOX2. The Mut− phenotype cannot grow on methanol as a single carbon source. We assessed the Mut− phenotype for secreted recombinant protein production. The methanol inducible AOX1 promoter (PAOX1) was active in the Mut− phenotype and showed adequate eGFP fluorescence levels and protein yields (YP/X) in small‐scale screenings. Different bioreactor cultivation scenarios with methanol excess concentrations were tested using PAOX1HSA and PAOX1vHH expression constructs. Scenario B comprising a glucose‐methanol phase and a 72‐hr‐long methanol only phase was the best performing, producing 531 mg/L HSA and 1631 mg/L vHH. 61% of the HSA was produced in the methanol only phase where no biomass growth was observed, representing a special case of growth independent production. By using the Mut− phenotype, the oxygen demand, heat output, and specific methanol uptake (qmethanol) in the methanol phase were reduced by more than 80% compared with the MutS phenotype. The highlighted improved process parameters coupled with growth independent protein production are overlooked benefits of the Mut− strain for current and future applications in the field of recombinant protein production.

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Section: Discussionmentioning

confidence: 99%

Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications

Zavec

Gasser

Mattanovich

2020

Biotech & Bioengineering

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“…Recombinant HSA has been produced in Hansenula polymorpha [11], Kluyveromyces lactis [12], Saccharomyces cerevisiae [13], rice [14], cattle [15], and mammalian cell lines [16]. Among the microbial systems, P. pastoris is considered to be the most promising platform [17]. One of the major challenges in this system has been the presence of contaminating proteins and instability of the secreted HSA.…”

Section: Introductionmentioning

confidence: 99%

Statistically Designed Medium Reveals Interactions between Metabolism and Genetic Information Processing for Production of Stable Human Serum Albumin in Pichia pastoris

Maity¹,

Mishra²

2019

Biomolecules

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Human serum albumin (HSA), sourced from human serum, has been an important therapeutic protein for several decades. Pichia pastoris is strongly considered as an expression platform, but proteolytic degradation of recombinant HSA in the culture filtrate remains a major bottleneck for use of this system. In this study, we have reported the development of a medium that minimized proteolytic degradation across different copy number constructs. A synthetic codon-optimized copy of HSA was cloned downstream of α–factor secretory signal sequence and expressed in P. pastoris under the control of Alcohol oxidase 1 promoter. A two-copy expression cassette was also prepared. Culture conditions and medium components were identified and optimized using statistical tools to develop a medium that supported stable production of HSA. Comparative analysis of transcriptome data obtained by cultivation on optimized and unoptimized medium indicated upregulation of genes involved in methanol metabolism, alternate nitrogen assimilation, and DNA transcription, whereas enzymes of translation and secretion were downregulated. Several new genes were identified that could serve as possible targets for strain engineering of this yeast.

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“…HSA is a 66.5 kDa protein with 35 cysteinyl residues forming 17 disulfide bridges plus one free thiolate (Kobayashi, ). Due to the high demand globally, research regarding yield optimization is ongoing, for example, recently codon optimization was explored to improve the production (Zhu et al, ). The ability to use a CFPS platform to screen for optimized variants such as these could increase the efficiency of using P. pastoris as a production host.…”

Section: Resultsmentioning

confidence: 99%

Biosensor‐assisted engineering of a high‐yield Pichia pastoris cell‐free protein synthesis platform

Polizzi

2019

Biotech & Bioengineering

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Cell‐free protein synthesis (CFPS) has recently undergone a resurgence partly due to the proliferation of synthetic biology. The variety of hosts used for cell‐free extract production has increased, which harnesses the diversity of cellular biosynthetic, protein folding, and posttranslational modification capabilities available. Here we describe a CFPS platform derived from Pichia pastoris, a popular recombinant protein expression host both in academia and the biopharmaceutical industry. A novel ribosome biosensor was developed to optimize the cell extract harvest time. Using this biosensor, we identified a potential bottleneck in ribosome content. Therefore, we undertook strain engineering to overexpress global regulators of ribosome biogenesis to increase in vitro protein production. CFPS extracts from the strain overexpressing FHL1 had a three‐fold increase in recombinant protein yield compared with those from the wild‐type X33 strain. Furthermore, our novel CFPS platform can produce complex therapeutic proteins, as exemplified by the production of human serum albumin to a final yield of 48.1 μg ml −1. Therefore, this study not only adds to the growing number of CFPS systems from diverse organisms but also provides a blueprint for rapidly engineering new strains with increased productivity in vitro that could be applied to other organisms.

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High level expression and purification of recombinant human serum albumin in Pichia pastoris

Cited by 47 publications

References 31 publications

Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications

Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications

Statistically Designed Medium Reveals Interactions between Metabolism and Genetic Information Processing for Production of Stable Human Serum Albumin in Pichia pastoris

Biosensor‐assisted engineering of a high‐yield Pichia pastoris cell‐free protein synthesis platform

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